A. Field of the Invention
The invention relates to a method and apparatus for improving mass spectrometry analysis of samples. Specifically, the invention relates to a method and apparatus for mass spectrometry analysis which allows for more precise alignment of a laser with samples, as well as for selection of impingement point(s) on each sample depending on the crystalline structure and other characteristics of the sample, to improve the quality of the data collected by a mass spectrometer.
B. Background Information
Mass spectrometry devices measure the molecular mass of a molecule by measuring the molecule""s flight path through a set of magnetic and electric fields. Such devices are well known and are widely used in the field of bio-molecular research. In proteomics research, for example, mass spectrometry is used to identify proteins.
Proteins are typically separated from one another by electrophoresis, such as the techniques described and claimed U.S. Pat. No. 5,993,627 to Anderson et. al. (hereinafter referred to as the Anderson et. al. patent), which is incorporated herein by reference in its entirety. For instance, as set forth in the Anderson patent, a tissue sample is first subjected to a first dimension electrophoresis process where groups of proteins are separated linearly within a tubular gel filled column. The first dimension separation of proteins is then inserted along an edge of a flat planar gel slab and subjected to a second dimension of electrophoresis, thereby generating a two dimensional pattern of spots formed by clusters of proteins that have moved to respective iso-electric focusing points. Thereafter, selected proteins are excised from the second dimension gel slab for further study. The selected excised spots are next prepared for analysis using, for instance, mass spectrometry.
An increasingly popular technique for studying biological molecules is the use of a matrix-assisted laser desorption ionization (MALDI) mass spectrometry apparatus wherein a biological sample such as an above-referenced excised spot is embedded in a volatile matrix which is subsequently vaporized by an intense laser emission. One such MALDI mass spectrometry apparatus is a MALDI-TOF apparatus (TOF is time-of-flight spectrometry). In the field of proteomics, mass spectrometry, and in particular, MALDI-TOF techniques are used to determine the molecular weight of peptides produced by digestion of isolated proteins. One such MALDI-TOF apparatus is VOYAGER DE STR Biospectrometry Workstation manufactured and sold by APPLIED BIOSYSTEMS.
The drawbacks of conventional methods for analyzing samples using mass spectrometry such as in proteomics research will become apparent from the following description of a conventional MALDI-TOF apparatus. FIG. 1 depicts a generic MALDI-TOF apparatus that includes a frame 1 that supports the electronic and computer equipment necessary to control a laser 5. The laser 5 is aimed at a fixed location in a positioning mechanism 10. The positioning mechanism 10 includes means (not shown) for positioning a sample in the line of fire of the laser 5. Typically, in a MALDI-TOF apparatus, the laser is fixed in place and the sample is moved into position for analysis.
The MALDI-TOF apparatus comprises a small removable sample plate 15, shown in FIGS. 2 and 3, that fits into the positioning mechanism 10. Typically, the sample plate 15 is insertable into a slot 20 in the positioning mechanism 10 of the MALDI-TOF apparatus and is thereafter held in a specific orientation within the positioning mechanism 10 for sample analysis. The sample plate 15 typically holds a plurality of discrete samples 16 on one surface thereof, with the samples 16 being spaced apart from one another, as shown in FIG. 3. The sample plate 15 includes guide members 15a, guide holes 15b and alignment pin 15d that are used by corresponding members (not shown) within the positioning mechanism 10.
The MALDI-TOF apparatus generally comprises a camera (not shown) in the positioning mechanism 10, which includes the sample plate 15 in its field of view, as well as the video monitor 25 depicted in FIG. 1. Thus, the MALDI-TOF apparatus can generate an analog output corresponding to the field of view to generate a display of the sample plate 15. Using the display, an array of Cartesian coordinates (X,Y) can be generated which corresponds to respective target sample areas on the sample plate 15. The sample plate 15 can then be moved automatically with respect to the line of fire of the laser 5 using these coordinates.
The samples 16 are loaded onto the sample plate 15 by a separate device or robotic apparatus that is typically manufactured and sold with each specific mass spectrometry apparatus. The robotic apparatus includes a recess that retains the sample plate 15 in position for sample loading, a first arm that moves back and forth along an X axis, and a second arm that moves along a Y axis defined along the length of the first arm. The second arm supports a pipette tip that is used to spot samples on the sample plate 15 as it is moved by the first and second arms.
Typically, an array of samples 16 are spotted on the sample plate 15 at predetermined locations, as depicted in FIG. 3. After the array of samples 16 are loaded onto the sample plate 15, the sample plate 15 is inserted into the slot 20 of the MALDI apparatus. Using the imaging system provided by the computer as indicated at 25, which is focused on the sample plate 15 within the MALDI apparatus, in combination with the positioning mechanism 10, the laser beam from the laser 5 can be aimed, one by one, at the sample(s) on the sample plate 15 in accordance with the array of coordinates.
In accordance with conventional methods for mass spectrometry analysis, the locations of the samples 16 are pre-programmed into the computer that controls the MALDI-TOF apparatus so that during the analysis of the samples, the positioning mechanism 10 automatically repositions the sample plate 15 into the line of fire of the laser 5. For example, a user enters via a mouse, keyboard or other input device an array of X-Y coordinates corresponding to sample positions on a sample plate. Thus, if any of the samples 16 on the sample plate 15 were not properly deposited in the target positions by the robotic apparatus, the laser 5 is not likely to hit those samples. More specifically, on the sample plate 15 depicted in FIG. 3, a 10xc3x9710 array of samples is positioned on the upper surface at spaced apart intervals. The positioning mechanism 10 moves into a target position with respect to centers of the desired or target location of each sample or spot. The desired location of each spot assumes that center of each of the spots in the 10xc3x9710 array is constant and therefore coincides with the centers 20 of the target areas 18, as depicted in FIG. 4.
Unfortunately, there are several shortcomings associated with the above-described robotic apparatus. Although the positioning mechanism 10 within the MALDI apparatus has positional accuracy with respect to movement of the sample plate 15, the robotic apparatus typically sold with a MALDI apparatus is not as precise with respect to accurate spotting or depositing of samples on the sample plate 15. Specifically, the spots 16 in a 10xc3x9710 array of samples are not centered on the desired center 20 targeted by the positioning mechanism 10. The array of 10xc3x9710 samples may have some samples (e.g., the sample 16a in FIG. 4) that are substantially accurately centered, and other samples (e.g., samples 16b and 16c) that are off center by as much as half the width of the sample. In addition, the crystalline structures of the samples can affect the manner in which they are deposited on the sample plate and therefore cause a certain degree of offset from the actual area of deposit for a sample and the target area for the sample plate.
During mass spectrometry analysis, the laser 5 is operated to impinge approximately five or six pre-determined locations relative to the pre-programmed centers 20 of each of the sample positions 18 on the sample plate 15 (e.g., points at predetermined positions on a circle surrounding the center 20 of a sample position). Since each impingement can be approximately one minute in duration, and sample plates can have arrays on the order of 100 to 144 samples, the mass spectrometry apparatus typically requires several hours to analyze an array of samples on the sample plate 15. If the laser does not impinge samples due to the afore-mentioned inaccuracies in depositing the samples on the sample plate, most of the data collected during this time-consuming process can be worthless.
Since the yield of useful output data from mass spectrometry equipment per each lengthy analysis period is relatively low, research laboratories have attempted to increase the data yield when collecting data during a single analysis period by using several mass spectrometers operating in parallel with respective sample plates. Mass spectrometers, however, are very expensive. Acquiring and operating several mass spectrometers at one time in an attempt to collect more data increases research costs considerably. Furthermore, a statistical average of as much as 50% of the data collected by the multiple mass spectrometers is essentially useless for the reasons stated above (i.e., from laser emissions that did not actually impinge samples 16). Accordingly, a need exists for an improved mass spectrometry analysis system that is cost effective and yields more reliable data.
In accordance with the present invention, a system is provided for precisely aligning the laser of a mass spectrometry apparatus with the samples on a sample plate to obtain more accurate sample data for improved mass spectrometry analysis.
In accordance with another aspect of the present invention, the system employs images as feedback to provide value-added direction to the positioning mechanism in the mass spectrometer apparatus.
In accordance with another aspect of the present invention, the system employs frame grabbing and image processing software to analyze images of individual samples and to select areas for each sample at which to aim a laser for mass spectrometry analysis, depending on the characteristics of that sample.